1. Field of the Invention
The present invention relates to a lymphoma cell line capable of producing large quantities of Kaposi's sarcoma-associated herpes virus (KSHV or HHV-8), which are substantially free from human immunodeficiency virus (HIV), Cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). This invention also relates to the purified virus produced thereby, and to methods for establishing HHV-8 producing cell lines, and for producing large quantities of the virus, as well as a method and kit for detecting HHV-8 infection.
2. Description of the Background
Kaposi's sarcoma (KS) is a rare neoplasm of multi focal origin characterized by red-purple to blue-brown lesions of the skin. Cell proliferation occurs initially in the skin, eventually spreading to other body sites, in particular to the lower extremities. Lymphatic involvement is not unusual in KS patents, and may be present as a lymphadenopathy. Kaposi's sarcoma is the most frequent neoplastic manifestation of HIV infection, and is used as one of the criteria to decide whether an HIV-infected individual is defined as having Acquired Immunodeficiency Syndrome (AIDS).
Four different epidemiologic forms of KS have been described: sporadic or classic KS, endemic KS, KS encountered among transplant recipients receiving immunosuppresive therapies, and KS prevalent among patients with human immunodeficiency virus (HIV) infection. The "classic" form of KS was described over a century ago in predominantly elderly men of Mediterranean and Jewish descent. Men are affected by this form of KS 10 to 15 times more often than women, and those affected are typically in their 60s or older, and have an average survival time of approximately 10 years. The "endemic" form of KS has been recognized in certain geographic regions of Central Africa. This is a neoplasm which also affects men more frequently than women, is generally more aggressive than classic KS, and involves the lymph nodes and viscera, as well. A marked increase in the form of KS encountered in patients receiving immunosuppressive therapy, was mostly found in hepatic and renal transplant patients. AIDS patients have a probability of about 40% of developing cancer, especially Kaposi's sarcoma and/or non-Hodgkin's lymphoma. Kaposi's sarcoma has, additionally, been associated with lymphoid cancer in patients both with and without AIDS.
Epidemiologic studies conducted with classic KS, endemic KS, and transplant patients suggest that both the infectious agent and the immune status of the individual are of significance in acquiring KS. The unidentified infectious agent, presumably the causative agent, has been referred to as Kaposi sarcoma-associated herpes virus (KSHV) and human herpes virus-8 (HHV-8), interchangeably. Two novel DNA fragments were found in 90% of KS lesions associated with AIDS. The isolation and identification of these DNA fragments from the KS lesions of an AIDS patient, designated KS330 and KS631, suggested the involvement of an infectious agent. The base sequences of the two DNA fragments, and their flanking sequences were shown to have significant homology with two known herpes viruses: herpes virus saimiri and Epstein-Barr Virus (EBV). The latter two viruses belong to the gammaherpesvirinae subfamily, whose members have the ability to replicate in lymphoblastoid cells. Of all members of the subclass, EBV is the best studied. EBV has been shown to induce latent infection of peripheral blood lymphocytes in its natural host, and to immortalize lymphocytes in vitro, thereby causing the development of malignant lymphomas such as endemic Burkitt's lymphomas, AIDS-related non-Hodgkin's lymphomas and lymphoproliferative disorders which occur after transplantation. A subset of non-Hodgkin's lymphomas, referred to as body cavity-based lymphomas BCBLs, or primary effusion lymphomas (PELs), present unique clinical, morphologic, immunophenotypic, and molecular genetic characteristics. BCBLs, for instance, grow mainly in the pleural, pericardial, and abdominal cavities, usually without an identifiable tumor mass. The cytomorphologic features of BCBLs appear to bridge large-cell immunoblastic and anaplastic large-cell lymphomas. Their cells are large and possess moderate to abundant amphophilic to deeply basophilic cytoplasm, and large, round to ovoid nuclei containing one or more large nucleoli. The lymphomas have indeterminate (non-B, non-T cell) immunophenotypes, and commonly express CD45 in the absence of other B or T cell lineage-restricted antigens. At the molecular level, the lymphomas are characterized by a B-cell genotype, as determined by clonal immunoglobulin gene rearrangements, and the absence of c-myc gene rearrangement.
As the numbers of reported AIDS cases increased, a concomitant rise in the incidence of KS was observed. This increase in KS has led to an increased effort to determine the pathogenesis of the neoplasm. The unavailability of a model system, however, has hindered these efforts.
The noticeable structural homology between EBV and KSHV has led to the hypothesis that KSHV might also be a transforming agent. In addition, KSHV/HHV-8 sequences were demonstrated in AIDS-related lymphomas, even in the absence of KS symptoms, but could not be detected in most non-AIDS-related lymphoid neoplasms. Since no source of HHV-8 free of other viruses was available, all cell lines established from neoplasms obtained from the HIV-infected individuals failed to provide, and could not be used as, a model system for KSHV/HHV-8, due to co-infection by other viruses, particularly EBV.
Herpes viruses as a group establish latent infections for the entire lifetime of their host. Their DNA genomes are relatively large, 100-250 kb, and may exist extra chromosomally in latently infected cells. The activation of an herpes virus results in viral replication, and eventually in cell lysis, with the viral copy number increasing substantially from the latent to the lytic infection stages. The latent stages of HSV in general, and KSHV in particular, infection produce no symptoms. The infected individuals, however, are still capable of transmitting the virus, and infecting others. Thus, although the KSHV/HHV-8 is present in the blood of seemingly healthy, yet infected, subjects, at present there are no acceptable methods to monitor the blood supply for HHV-8 infection.
Clearly, thus, up to the present time, the isolation and characterization of KSHV has been hampered because all cell sources have been co-infected with EBV, CMV, and/or HIV. Thus, the availability of a ready, abundant, and uncontaminated source of KSHV/HHV-8 would permit the systematic monitoring of blood bank stocks as well as of new blood donors to avoid the spread of the virus. In addition, it would permit the development of specific antibodies, and to conduct further pathogenicity studies.